Another “ome” has joined the biological universe. The glycoproteome has become the object of intense study. While scientists are hunting for novel tools to facilitate its analysis, assay developers are focusing on high-throughput analytical instrumentation.

More than half of all proteins that have been characterized are glycoproteins, that is, polypeptide chains with glycans or sugars covalently attached to the peptide backbone. The carbohydrate components of glycoproteins perform critical biological functions in protein sorting, immune and receptor recognition, inflammation, and pathogenicity. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAc, and NANA.

Glycans and glycoslyation patterns assume particular importance as they significantly modify the activity of recombinantly produced therapeutic proteins. Chief among concerns are the occurrence of adverse events secondary to immune responses to therapeutic glycoproteins. These include hypersensitivity reactions such as anaphylaxis, rash, fever, and kidney problems.

Tapping LC/MS

Certain characteristics of glycoproteins, though, make them especially difficult to analyze. Beyond characterization and quantitation of individual glycans, determining structures and structure/function relationships is complicated by the occurrence of the glycans as mixtures of closely related structures, with complex branching patterns and labile bonds.

Nevertheless, modern mass spectrometry (MS) techniques, coupled with improvements in sample preparation and separation methods, are reportedly enabling this task to be accomplished at sensitivity levels that approach those for proteins and peptides.

Amgen’s Gregory Flynn, Ph.D., scientific director, process and product development, told GEN that there are two main approaches to analyzing antibody glycans: one based on enzymatic glycan release followed by derivatization with fluroescent labels, and one based on peptide mapping.

“Both of these can be made MS compatible to allow characterization and in some cases quantification.” He also noted that higher-throughput technologies using UltraPerformance Liquid Chromatography® (UPLC®) or microchip CE have also been developed. Most current assays require significant sample prep, though, Dr. Flynn added. “For real-time analysis, such as needed for in-line control or monitoring, more sensitive assays based on whole-mass analysis might be preferred.”

Commercial interest in a new high-throughput method for automated, quantitative analysis of N-linked sugar structures attached to proteins is indicative of a growing industry-wide focus on glycan analysis.

Pauline Rudd of the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin and professor of glycobiology at University College, Dublin, and other researchers from Boston’s Brigham and Women’s Hospital and Agilent Technologies, teamed up to develop a 2-D LC separation platform for glycan research. Based on Agilent’s HPLC chip technology, the scientists say the platform enabled high throughput of low-femtomoles analysis of N-linked sugars released from glycoproteins.

Last year Waters and the NIBRT announced a collaboration to create the world’s first database for glycan analysis by UPLC. Expected to be available this year, NIBRT will develop, maintain, and license the database, and Waters and NIBRT will co-market it worldwide.

Microarray Innovation

A real game changer for glycomics, though, may be the introduction of carbohydrate microarrays to support high-throughput analysis of protein-glycan interactions. These arrays consist of diverse glycans densely attached to a solid surface in an orderly arrangement.

The microarrays would allow simultaneous analysis of multiple glycan-protein interactions using small amounts of carbohydrate samples. While work is still in progress, these microarrays have the potential to supplant ELISA-based microplate array for routine screening of glycan-binding protein.

Since most carbohydrate-binding proteins achieve tight binding through formation of multivalent complexes, both ligand structure and presentation contribute to recognition. Also, since there are many potential combinations of structure, spacing, and orientation to consider, and the optimal one cannot be predicted, high-throughput approaches for analyzing carbohydrate-protein interactions and designing inhibitors are appealing.

In 2010, NIH scientists reported development of a strategy to vary neoglycoprotein density on a surface of a glycan array. This feature was combined with variations in glycan structure and density to produce an array with approximately 600 combinations of glycan structure and presentation. The unique array platform allows the investigators to distinguish between different types of multivalent complexes on the array surface.

The investigators used the technology to rapidly identify potent multivalent inhibitors of Pseudomonas aeruginosa lectin I (PA-IL), a key protein involved in opportunistic infections of P. aeruginosa, and mouse macrophage galactose-type lectin (mMGL-2), a protein expressed on antigen-presenting cells that may be useful as a vaccine-targeting receptor. An advantage of the approach is that structural information about the lectin receptor was not required to obtain a multivalent inhibitor/probe.

The Consortium for Functional Glycomics (CFG), originally funded by the National Institute of General Medical Sciences (NIGMS), provides multiple resources for researchers working in glycomics. Its mission, it says, is to provide a networking forum and glycomics resources that enable investigators to reveal functions of glycans and glycan-binding proteins that impact human health and disease.

The CFG offers glycan microarray screening services, a reagent bank, and free access to its extensive data repositories and molecule databases. In 2006, the CFG teamed up with the Nature Publishing Group (NPG) to create the Functional Glycomics Gateway, which encompassed the existing CFG website and databases and the Functional Glycomics Update from NPG. As of September 1, the Functional Glycomics Gateway became supported solely by the CFG.

One of the resources provided by the CFG is its printed mammalian glycan microarrays that allow glycan-binding proteins to be screened against a wide variety of glycans for binding specificity. To generate the printed array, a growing library of natural and synthetic mammalian glycans with amino linkers is printed onto N-hydroxysuccinimide (NHS)-activated glass microscope slides forming covalent amide linkages. The current mammalian array (version 5.0) has 611 glycan targets.

The discovery that the CFG printed mammalian glycan array can be used to define the specificity of glycan-specific antibodies in human sera opened up the possibility of evaluating the potential of glycan-specific antibodies as biomarkers for cancer. To this end, the CFG assessed the specificity of several commercial and noncommercial glycan-specific mAbs to define their cross-reactivity with other epitopes.

As novel analytical tools that make glycoprotein analysis more accessible are becoming available, the field of functional glycomics is taking shape and growing in prominence among the other omics fields.

Researchers are also refining and extending conventional tools to reveal how glycans behave and work in a functional context. All these technologies will be needed to support both basic research designed to advance the science of glycomics and characterization of the human glycoproteome.

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